Microbial oxidation of polynuclear aromatic hydrocarbons



United States Patent 3,318,781 MICROBIAL OXIDATION OF POLYNUCLEAR AROMATIC HYDROCARBONS Ira D. Hill, Wilmington, Del., assignor to Sun 0i] Company. Philadelphia, Pa., a corporation of New Jersey No Drawing. Filed Sept. 1, 1964, Ser. No. 393,717

16 Claims. (Cl. 195-28) This invention relates to the oxidative ring-splitting of polynuclear aromatic hydrocarbons by microbial means to form the corresponding hydroxy acids. More particularly, it relates to an improved microbiological method for converting naphthalene, phenanthrene, and anthracene to their respective aromatic hydroxy acids in high yields by use of certain combinations of substrates.

Methods for converting polynuclear aromatic hydrocarbons to their corresponding hydroxy acids by microbial means have been reported by many prior art workers. Thus, for example, Arnaudi et al. have reviewed several such methods in Sci. Repts; dist. Super. Sanita, 1, 378-386 (1961), wherein naphthalene, phenanth'rene, and anthracene are reported to have converted to salicylic acid and intermediate hydroxy naphthoic acids respectively. However, in each instance, these processes have been characterized by very low orders of yields, generally about 10 to 100 mg. per liter or lower, which yields have rendered these processes commercially unattractive. On the other hand, the desirability of converting such relatively inexpensive aromatic hydrocarbons to their more valuable hydroxy acids by low cost microbiological means is obvious, and it is thus an object of this invention to provide a commercially feasible fermentation method for producing these oxidation products in high yields.

In accordance with the present invention, it has now been found that this and other objects can be accomplished by carrying out the fermentation of polynuclear aromatic hydrocarbon substrates with suitable oxidative ring-splitting microorganisms in the presence of a carbohydrate co-substrate which is capable of supporting the growth of said oxidative microorganism. By conducting the oxidation of the aromatic hydrocarbons in this fashion it has been discovered that while a very material increase in the growth of the cells takes place in the presence of the carbohydrate co-substrate over the normal rate of growth experienced with aromatic hydrocarbons as the sole carbon source, nevertheless, this growth surprisingly is not at the expense of the oxidation of the hydrocarbons, as would normally be expected. On the contrary, despite the microorganisms preference for the such more readily assimilable carbohydrates, the present invention results in the yield of a measurably greater amount of oxidation products than had heretofore been possible from prior methods, e.g. yields of up to 5 gms. per liter of broth. Thus, it would appear that the microorganism, while growing at the expense of the carbohydrate co-substrate, nevertheless still utilizes the aromatic hydrocarbon as a source of energy with the result that the aromatic ring is split and simultaneously oxidized to form the desired hydroxy acids.

The aromatic hydrocarbons and substituted aromatic hydrocarbons oxidized in accordance with the present process are those containing at least two condensed rings,

such as naphthalene, anthracene, phenanthrene or the like, including the halo and nitro derivatives thereof, wherein at least one of said rings is free from any such substituents. Thus, for example, when naphthalene, anthracene, and phenanthrene are oxidized in accordance with the present process, there are obtained salicylic acid, 2-hydroxy-3-naphthoic acid, and 1-hydroxy-2-naphthoic acid respectively.

3,318,781 Patented May 9, 1967 ice The carbohydrate substrates employed in this process include any of the known sugars and starches or mixtures containing them which are capable of supporting the growth of the selected ring-splitting oxidative microorganism, as for example, molasses, corn syrup, hydrolyzed starch, and the like, and preferably glucose.

The fermentation medium must contain, in addition to the foregoing substrates, a mineral salts medium on which the organism can grow, and should, in any event, contain an available source of nitrate, phosphate, sulfate, ferric, cupric and like ions as well as other trace metal ions known in the art. Examples of suitable sources of these ions are such compounds as ammonium nitrate, potassium diphosphate, magnesium sulfate and the like diluted with a sufficient amount of water and adjusted to a pH of about 5.5 to 8.5 and preferably a pH of from about 7.2 to 7.6. In addition to the foregoing nutrients, it has been found that the rate of production of hydroxy naphthoic acids can be further increased somewhat if such growth-stimulating materials as yeast extract (cg. Difco), at concentrations of about 0.001 gm. per liter are added and preferably from .0005 to .005 gm. Other suit-able growth-stimulating materials which may also be used are such substances as pure vitamin mixtures, distillers solubles, corn steep liquor or the like.

The microorganisms which may be satisfactorily employed in this process are any of those capable of ringsplitting and oxidizing aromatic hydrocarbons while simultaneously utilizing carbohydrates to support their growth. One such organism which has been found to be particularly effective in this regard is a newly discovered microorganism designated as Corynebacterium nov. sp. (H-l), a culture of which has been deposited with the American Type Culture Collection in Washington, DC, under the number ATCC 15,570. This microorganism, which is gram positive, motile, with short rods and demonstrates a marked ability to use carbohydrates as a co-substrate, was isolated fro-m the soil in Marcus Hook, Pa., by a naphthalene enrichment technique whereby 5 gm. of soil and 1 gm. of solid naphthalene were added to ml. of mineral salts medium (described below) and shaken at 30 C. for two days. The organism was then isolated by plating the resulting enriched broth on the same mineral salts medium which was solidified with 2% agar. This organism was found to have the following cultural and physiological characteristics which identified and distinguished it from other microorganisms:

STAINlN G CHARACTERlSTlCS Age, hours: Gram 24 Gm;+ 48 Gm+ 72 Gm+ and Gm 168 Gm- CELL MORPHOLOGY Form Very short rod. Motility Sims mediamotile (apparent in 4 hours). Size:

24 hours 0.4 to 0.6 by 1.0 to 1.2 microns. 48 hours 0.4 to 0.6 by 1.0 to 1.7 microns. 72 hours 0.4 to 0.8 by 0.4 to 0.8 microns.

AGAR COLONIES Age 72 hours. Form Circular. Elevation Convex. Surface Smooth. Margin Entire. Chromogenesis Tan, irridescent.

AGAR STROKE Age 72 hours. Form Filifor'm. Consistency Butyrous. Chromogenesis Tan. Scant Growth.

NUTRIENT BROTH Surface Growth Ring. Subsurface Growth Turbid. Amount Abundant. Sediment Viscid.

GELATIN STAB Liquefaction None. Growth None.

POTATO SLA-NT Age 72 hours. Chromogenesis Yellow-tan. Consistency Viscid.

POTATO DEXTROSE COLONIES Age 72 hours. Chromogenesis White, good growth. Consistency Very Viscid.

GLUCOSE AGAR Age 72 hours. Chromogenesis White, good growth. Consistency Butyrous. Good Growth.

ACTION ON SUGARS Maltrose No acid, no gas,

no growth. Sorbitol Do. Dextrose Do. Mannitol D0. Lactose D0. L-Arabinose Do. Sacchrose Do. Levulose Alkaline, no gas,

slight growth. Inositol No acid, no gas,

growth.

ACTION ON MILK Reaction:

No change First week. Cogulation and reduction Third week. Peptonizations Fourth week.

No nitrates from nitrates Hydrogen sulfide not produced Indole not produced No phenol utilization Naphthalene utilized Grease not produced Starch not hydrolyzed Utilizes ammonium salts as a nitrogen source The process of this invention is desirably carried out at a temperature of from about C. to 40 C., and

preferably from 28 C. to 32 C. under aerobic conditions with agitation. The necessary oxygen may be supplied either by bubbling air through the fermentation medium or by agitating the broth sufficiently while exposed to the atmosphere. The pH of the fermentation broth, as noted above, should be kept at near neutrality or slightly alkaline, preferably at a pH of 5.5 to 8.5, depending upon the nature of the organism and nutrient medium employed.

The polynuclear aromatic hydrocarbon and carbohydrate co-substrate are desirably added to the nutrient mineral salts medium prior to the inoculation with a suitable microorganism; but in any event, it is essential that the fermentation be carried out in the simultaneous presence of both substrates. The reason for this is that it has been found that surprisingly, when the organism is first grown solely in the presence of, for example, glucose, followed by the later introduction of the aromatic hydrocarbon, no oxidation products whatever are recovered. Therefore, in practice, both substrates should be present in the fermentation medium in suitable concentrations during the growth phase of the organism at the same time. In adding the aromatic hydrocarbon to the medium, the total quantity of hydrocarbon employed is not critical but must always be in excess of the amounts required to saturate the total nutrient medium. The carbohydrate co-subst'rate should constitute from 0.05% to 0.5% of the total fermentation broth and preferably from 0.1% to 0.2%. Both the aromatic hydrocarbon and carbohydrate may be added in dry powdered form. Alternatively, the hydrocarbons may be introduced as crude mixtures obtained from naphthalenic cuts of crude oil. In either case, it is essential that the selected substrates first be sterilized before introduction into the fermentation medium in order to avoid contamination.

For optimum yields of fermentation product, the fermentation should be carried out for a period of from 24 to 168 hours, and preferably from 48 to hours. However, since the microorganisms have atendency to metabolize the desired end products over long periods of time, it is desirable that a periodic assay be maintained to determine the optimum recovery period. Severai methods are known for determining the presence of salicylic or hydroxy napthoic acids. For example, one chemical method for assaying salicylic acid is as follows:

A naphthalene shake flask which has produced salicylic acid is allowed to continue shaking until no more salicylic acid can be observed by FeCl spot test even after extensive liquid-liquid extraction. To this fermentation beer is added a weighed amount of salicylic acid and the pH adjusted to 6.5 where upon the salicylic acid dissolves. A 0.5 ml. sample of this beer is then placed in a test tube and a duplicate amount is placed in a similar test tube as a reference blank. To the first test tube is added 0.5 ml. of a 1% HNO solution while 1.0 ml. of a 1% HNO solution is added to the blank tube. Thereafter, 0.5 ml. of a 5% Fe(NO reagent is added to the first tube. The color tube and the blank tube are then shaken vigorously and 2.5 ml. of water is added to each of the tubes. If the color in the tubes appears too dark to read in a spectrophotometer, the tubes should be diluted by adding water in multiples of 4.0 ml. The solutions are then filtered through cotton plugs and read on a Beckman-DKZ spectrophotometer for absorbance at 525 m After correction for the reading of a blank containing no salicylic acid, a straight line relationship is found to exist between color absorbance and concentration of salicylic acid over a range of .05 to 1.0 mg./ml. The salicylic acid content of the unknown filtrate is calculated from a pre-prepared calibration curve plotting absorbance at 525 m against mgs. per ml. of test material. The actual amount of salicylic acid present is then determined by the following relationship:

(1) Find the mg./test on the graph corresponding to the absorbance at 525 mp.

ml. beer added Similarly, the amount of l-hydroxy-Z-naphthoic acid or 3-hydroxy-2-naphthoic acid present in the broth may be measured by the following assay method:

A 10 ml. sample of the fermentation beer which has been centrifuged free of cells and removing starting material is adjusted to a pH of 9-10 with a solution of sodium hydroxide. The solution is then extracted with 20 ml. of diethyl ether to remove any residual hydrocarbon starting material. The hydrocarbon-free solution is then acidified with concentrated hydrochloric acid to a pH of 1-2, and again extracted twice with 20 ml. of diethyl ether. The combined ether extracts are evaporated to dryness, the recovered solids dissolved in 10 ml. of absolute methanol, and appropriate dilutions of this methanol solution with additional methanol are read on a Beckman-DKZ spectrophotometer for absorbance corresponding to the respective hydroxy naphthoic acid. The number of rug/ml. of acid may then be calculated as follows:

Absorbance for l-hydroxy-Z-naphthoic acid is read at Then 'Mg./ ml.

absorbance dilution Absorbance for 2-OI-I-3-naphthoic acid is read at 235 m Then:

Mg./ ml.

absorban ce dilution The salicylic and hydroxy naphthoic acid fermentation products may conveniently be recovered from the final fermentation broth by a number of known methods. Thus, for example, salicyclic acid may readily be re covered by filtering or centrifuging the whole broth to remove the microorganism and other solids, then contacting the resulting liquor with activated carbon to yield a product which is 99% pure based on reagent grade salicylic acid. This recovery is, most conveniently achieved by adjusting the pH of the filtered or centrifuged broth to 7, contacting this broth with 3 weight percent (based on salicylic acid content) of activated carbon at 200 F., cooling and filtering the broth, and acidifying it with a mineral acid such as hydrochloric to a pH 1 to crystalize out 99% pure salicylic acid. This product may, ifdesired, be further purified by recrystallization from such solvents as acidified water.

Alternatively, salicylic acid may readily be recovered from filtered fermentation broth by neutralizing the broth, contacting it with the chloride form of a weakly basic anion exchange resin such as Amberlite-IR-45, and eluting the absorbed acid from the resin with acidified methanol. The salicylic acid is then conveniently recovered from the methanol solution by evaporating it to dryness.

These and similar processes may also be satisfactorily employed to recover the corresponding hydroxy naphthoic acids. However, when these products are recovered, they may, if desired, be further purified by recrystallization from a suitable organic solvent, such as hot benzene.

The following examples are given for purposes of illustration of the afore-described invention, but are not intended to limit the same.

Example 1 A series of fermentations were carried out in 500 ml. sterilized, dispo-plugged, fluted Erlenmeyer flasks, each containing ml. of a sterile mineral salts medium having the following composition:

Salts: Grams/ liter K HPO 10.0 KH PO 1.0 NH 'NO 2.0

FeSO4.7H O .001 C11SO .5H O .0002 H BO .00006 MnSO .H O ZnSO l-I O .n .0002 NH2MO4.2H2O CaCl .6H O .0011 NiCl .6H O .000005 The pH of the medium was adjusted to 7.4. To each flask was added 1 gm. of solid, sterile, naphthalene crystals. Where glucose was added, it was sterilized and added as a 10% solution after cooling.

Corynebacterium nov. sp. (ATCC 15,570), which had been previously grown on a plate of the above-described medium together with a 2% yeast extract (Difco) agar inverted over naphthalene, was washed off the plate with sterile Water and aliquots of the wash were used to inoculate the fermentation flasks.

The flasks were shaken at a room temperature of about 24-28 C. on a rotary shaker for periods ranging from about 20 to 112 hours. The high phosphate content of the mineral salts medium was suflicient to control the pH during the course of the experiment without any further adjustments. It will be understood, however, that a low phosphate content, as little as 10% of the amount normally added, may be employed in conjunction with an automatic pH control device.

During the fermentation period, samples of the fermentation broth were taken periodically and were centrifuged to remove bacterial cells and the like. The amount of salicylic acid in the clear supernatant fluid was determined on a spectrophotometer. The following results were obtained in grams/ liter:

Example 1 was repeated except that 0.01% yeast extract (Difco) was added aseptically to the cooled flasks with the following assay results, measured in grams/ liter:

Hours of Fermentation (Jo-substrate 20 hours 30 hours 40 hours 112 hours No glucose 0 1.36 1. 40 1.40 0.1% glucose 1. 72 2. 16 1. 20 0.70

Example 3 The maximal rate of production of l-hydroxy-Z- naphthoic acid, as determined by spectrophotometric assay methods was 0.1-0.2 gm./liter/ day with no glucose added, and a maximal rate of 1.9 gm./liter/day with the addition of 0.1% glucose.

The procedure of Example 2 was repeated, including the 0.01% yeast extract, except that pure anthracene (1.0 gm.) was substituted for naphthalene. By spectrophotometric assay, the following yields of 2-hydroxy-3- naphthoic acid were measured in grams/ liter:

1 Quantity too slight for quantitative assay.

It is thus demonstrated by the foregoing experiments that the use of a carbohydrate co-substrate in the oxidative ring-splitting of polynuclear aromatic hydrocarbons by microbial means surprisingly and unexpectedly increases the yield of the resulting hydroxy acids by as much as 100 fold over corresponding fermentations where no carbohydrate co-substrate is added. That such a result is unexpected is evident from the work of I. Monod reported in Recherches sur la croissance des Cultures Bacteriennes, Hermann, Paris (1941), as further explained by Gunsalus et al. in The Bacteria, 3, p. 610 et seq., Academic Press (1962). In that latter work it is reported that the addition of glucose to a fermentation has long been known to inhibit the formation of many enzymes, utilized by the organism in that fermentation, which inhibition is known as Monods diauxie effect. This effect has been manifested, for example, when glucose is added to the fermentation of a more diflicultly assimilated sugar. Invariably, the organism first attacks the more readily assimilated glucose exclusively, and then the more difiicultly assimilated sugar. From this observation it has been concluded by prior art workers that the presence of an easily assimilated substrate will prevent the use of a more difiicultly assimilated substrate until the first substrate is completely utilized. Accordingly, the utilization by a microorganism of a sugar for growth purposes while it is simultaneously oxidizing a diflicultly assimilated aromatic hydrocarbon where each of these steps when carried out in succession, produces no yield whatever, provide a wholly unexpected and advantageous result in the conversion of aromatic hydrocarbons to hydroxy acids.

The invention claimed is:

1. In the process for the oxidative ring-splitting of polynuclear aromatic hydrocarbons by microbial fermentation utilizing Corynebacterium nov. sp. ATCC 15,570 to form the corresponding hydroxy acids, the improvement which comprises conducting such an oxidative fermentation in the presence of a carbohydrate co-substrate in amount of at least 0.05%.

2. The process according to claim 1 wherein the carbohydrate is glucose.

3. The process according to claim 1 wherein the amount of carbohydrate used is in the range of 0.05% to about 1.0%.

4. The process according to claim 1 wherein the polynuclear aromatic hydrocarbon is naphthalene and the product is salicylic acid.

5. The process according to claim 4 wherein the amount of carbohydrate used is in the range of 0.05% to about 1.0%.

6. The process according to claim 4 wherein the carbohydrate is glucose.

7. The process according to claim 1 wherein the polynuclear aromatic hydrocarbon is phenanthrene and the product is l-hydroxy-Z-naphthoic acid.

8. The process according to claim 7 wherein the amount of carbohydrate used is in the range of 0.05% to about 1.0%. Y

9. The process according to claim 7 wherein the carbohydrate is glucose.

10. The process according to claim 1 wherein the polynuclear aromatic hydrocarbon is anthracene and the product is 2-hydroxy-3-naphthoic acid.

11. The process according to claim 10 wherein the amount of carbohydrate used is in the range of 0.05% to about 1.0%.

12. The process according to claim 10 wherein the carbohydrate is glucose.

13. The process which comprises contacting a polynuclear aromatic hydrocarbon selected from the group consisting of naphthalene, anthracene, phenanthrene and the halo and nitro derivatives thereof with Corynebacterium nov. sp. ATCC 15,570 in the presence of at least 0.05% of a carbohydrate co-substrate to form the corresponding hydroxy benzoic and hydroxy naphthoic acids.

14. The process according to claim 13 wherein the amount of carbohydrate used is in the range of 0.05% to about 1.0%.

15. The process according to claim 13 wherein the carbohydrate is glucose.

16. The process which comprises contacting a polynuclear aromatic hydrocarbon selected from the group consisting of naphthalene, anthracene, and phenanthrene with Corynebacterium nov. sp. (ATCC 15,570) in an aqueous nutrient under aerobic conditions in the presence of from 0.05% to about 1.0% of glucose to form the corresponding hydroxy benzoic and hydroxy naphthoic acids.

References Cited by the Examiner UNITED STATES PATENTS 3,183,169 5/1965 Brillaud 28 A. LOUIS MONACELL, Primary Examiner.

L. M. SHAPIRO, Assistant Examiner. 

1. IN THE PROCESS FOR THE OXIDATIVE RING-SPLITTING OF POLYNUCELAR AROMATIC HYDROCARBONS BY MICROBIAL FERMENTATION UTILIZING CORYNEBACTERIUM NOV. SP. ATCC 15,570 TO FORM THE CORRESPONDING HYDROXY ACIDS, THE IMPROVEMENT WHICH COMPRISE CONDUCTING SUCH AN OXIDATIVE FERMENTATION IN THE PRESENCE OF A CARBOHYDRATE CO-SUBSTRATE IN AMOUNT OF AT LEAST 0.05%. 